Project Summary: Lyme disease (LD) is the most common vector-borne disease in the northern hemisphere. The disease is caused by the spirochete Borrelia burgdorferi sensu lato (Bbsl), which spreads from a tick bite to the skin to different tissues, leading to arthritis, carditis, and neuroborreliosis. No effective prevention is currently available. Our goal is to understand the mechanisms of protective immunity to develop safe and potent prevention tools against human LD. Complement is an important innate defense mechanism in the blood that can be triggered by multiple pathways including the classical pathway, which is induced by antibody-antigen complexes, and the alternative pathway, which is triggered by the binding of the complement C3b protein with the microbial surface. The activation of complement results in a pore-forming complex, C5b-9, on the bacterial surface leading to lysis. In the absence of pathogens, complement is inhibited by complement regulators to avoid host cell damages. For example, Factor H (FH) specifically inhibits the alternative pathway. Spirochetes produce an outer surface protein, CspZ, widely present in Bbsl species that can be efficiently transmitted to mammalian hosts. CspZ is produced when bacteria enter these hosts and facilitates Bbsl dissemination by recruiting FH to its surface thus inhibiting complement-mediated killing. However, immunization of CspZ neither induces great levels of bactericidal antibodies nor does it protect mice from Bbsl colonization. One possibility is that CspZ?s protective epitopes are saturated by FH, which would not allow this protein to induce sufficient antibodies to efficiently eliminate Bbsl in vivo. We thus generated a CspZ-Y207A/Y211A mutant (CspZ-YA) that is deficient in FH-binding, leading to the exposure of the epitopes on this protein?s FH-binding sites. We found that CspZ-YA but not CspZ vaccination protects mice from Bbsl colonization via tick infection. We demonstrated that passive immunization of the antibodies from CspZ-YA- but not CspZ-immunized mice protects mice from Bbsl colonization. We observed that the former but not later antibodies block FH binding to CspZ. These findings suggest that CspZ- YA?s FH-binding site is exposed, which could induce protective antibodies to promote Bbsl clearance. Thus, the overall objective is to define the protective mechanisms of the CspZ-YA vaccine. We hypothesize that the protective antibodies from CspZ-YA vaccination block FH-dependent evasion of the alternative pathway and promote activation of the classical pathway, resulting in Bbsl killing. To test the hypothesis, we will 1) assess the role of the antibodies induced by CspZ-YA vaccination in promoting the activation of classical and alternative complement pathways, 2) define the mechanisms of the CspZ-YA-induced antibodies leading to Bbsl clearance. These studies will elucidate the mechanisms that allow CspZ-YA to function as an effective vaccine. Such mechanisms will build the foundation to further identify the protective epitopes of this antigen to identify potent monoclonal antibodies as LD prophylactic agents. Overall, this information will ultimately provide effective strategies to prevent Bbsl infection and reduce the burden of human LD.